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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarWorks@UMass Amherst University of Massachusetts Amherst ScholarWorks@UMass Amherst Masters Theses 1911 - February 2014 2007 A Molecular Phylogenetic Assessment of Pseudendoclonium Richard F. Mullins University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/theses Mullins, Richard F., "A Molecular Phylogenetic Assessment of Pseudendoclonium" (2007). Masters Theses 1911 - February 2014. 47. Retrieved from https://scholarworks.umass.edu/theses/47 This thesis is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Masters Theses 1911 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM A Thesis Presented by RICHARD F MULLINS, JR. Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE September 2007 Plant Biology Graduate Program A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM A Thesis Presented by RICHARD F. MULLINS, JR. Approved as to style and content by: __________________________________________ Daniel R. Cooley, Chair __________________________________________ Robert L. Wick, Member __________________________________________ Margaret A. Riley, Member ________________________________________ Elsbeth L. Walker, Director Plant Biology Graduate Program ACKNOWLEDGMENTS It is with deepest gratitude that I acknowledge the invaluable assistance and untiring support of my good friend and mentor Dr. Robert T. Wilce. Completion of this project would not have been possible without Bob’s persistent encouragement and insightful advice. His kindness and generosity have been of such magnitude that “thank you” doesn’t even begin to cover it. I thank Dr. Daniel R. Cooley for sharing his extensive practical and professional wisdom as my academic advisor. I am especially grateful to Dan for his efforts securing financial support for my studies and for laboratory supplies. I am grateful to Dr. Robert L. Wick for his participation as a thesis committee member and for his helpful advice. I thank Rob also for a solid introduction to the science of Plant Pathology, for providing teaching assistantships and for sharing laboratory equipment. I am fortunate to have Dr. Margaret A. Riley as a thesis committee member and as a teacher. I could not have effected the phylogenetic analyses that are of central importance to this thesis, without having taken her course in molecular evolution. I am most grateful that she has taken time from a very busy schedule to review this work. N. Ezekiel Nims has and deserves my sincere gratitude for his assistance with laboratory protocol. Zeke rescued me from the PCR doldrums. Milind Chavan has been most helpful with the details of PCR. I thank him for his time and the patience required in answering the countless questions of a novice. I thank the Plant Biology Graduate Program for their accommodation of a “non- traditional” student and for the opportunity to pursue a graduate degree. iii From my perspective, Susan Capistran is the most dedicated program director on this campus. Thank you so much for your assistance, patience and understanding. David Fill, a maintenance supervisor for Umass Dining Services, has provided me with part-time employment for the duration of my graduate career. As my boss, Dave has been most understanding and supportive of my graduate studies. He has patiently allowed scheduling changes necessary for the completion of this work. For this, I owe him much and offer my sincere gratitude. iv ABSTRACT A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM SEPTEMBER 2007 RICHARD F. MULLINS JR., B.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Daniel R. Cooley Pseudendoclonium was established in 1900 by N. Wille to include a crust- forming green microalga occurring near the high water line on jetties in Drobak, Norway. Ordinal and familial affiliation of the genus have remained uncertain due to a lack of distinguishing morphological characteristics and because molecular phylogenetic data have not been generated for the type species. Ribosomal SSU rDNA sequence data for Pseudendoclonium submarinum, the type species, are presented. Phylogenetic analysis of these data place Pseudendoclonium within the Ulvales. SSU rDNA sequence data from three additional species, Pseudendoclonium basiliense, Pseudendoclonium akinetum and Pseudendoclonium fucicola are included in the analyses and clearly support the hypothesis that Pseudendoclonium is polyphyletic. Based on the sequence data, P. submarinum and P. fucicola share ulvalean lineage, but these algae are not congeneric and P. fucicola must be removed from Pseudendoclonium. Sequence data support the classification of P. basiliense and P. akinetum as distinct species of a single genus. The close affiliation of these two species with Ulothrix and other Ulotrichalean genera, however, reveals their ordinal separation from P. submarinum. P. basiliense and P. akinetum must also be removed from Pseudendoclonium and require generic reassignment within the Ulotrichales. v TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...............................................................................................iii ABSTRACT...................................................................................................................... v LIST OF TABLES.........................................................................................................viii LIST OF FIGURES .........................................................................................................ix CHAPTER 1. THE GENUS PSEUDENDOCLONIUM .............................................................. 1 1.1 Introduction..................................................................................................... 1 1.2 Taxonomic History......................................................................................... 2 2. MATERIALS AND METHODS.......................................................................... 6 2.1 Isolation and Culture....................................................................................... 6 2.2 Microscopy ..................................................................................................... 7 2.3 DNA Extraction.............................................................................................. 8 2.4 DNA Amplification ........................................................................................ 9 2.4.1 PCR Primers.................................................................................. 9 2.5 Detection and Analysis................................................................................. 10 2.5.1 Purification.................................................................................. 10 2.5.2 Sequencing.................................................................................. 10 2.5.3 Sequencing Primers.................................................................... 11 2.5.4 Sequence Editing........................................................................ 11 2.6 Phylogenetic Analysis................................................................................... 12 3. RESULTS........................................................................................................... 15 3.1 General.......................................................................................................... 15 3.2 Partial-Sequence Analysis ............................................................................ 16 3.3 Full-Sequence Analysis ................................................................................ 18 4. DISCUSSION..................................................................................................... 20 vi 4.1 Generic.......................................................................................................... 20 4.2 Specific ......................................................................................................... 21 5. CONCLUSION................................................................................................... 27 5.1 Evolutionary Considerations......................................................................... 27 5.2 Specific Assignment ..................................................................................... 28 REFERENCES ............................................................................................................... 38 vii LIST OF TABLES Table page 1.1 The Genus Pseudendoclonium ...................................................................... 32 2.1 Composition of Modified von Stosch’s Enriched Seawater Medium .......... 32 2.2 Oligonucleotide Primers Used in this Study................................................. 33 3.1 SSU rDNA Sequence of Pseudendoclonium submarinum ........................... 34 viii LIST OF FIGURES Figure page 1.1 Growth Habit of P. submarinum in Liquid Culture...................................... 35 1.2 Scanning Electron Micrographs of Pseudendoclonium submarinum ...........35 3.1 Maximum Likelihood Phylogram Inferred from Partial 18s rDNA Sequences of 25 Green Algal Taxa Illustrating Ordinal Affiliation of Four Species of Pseudendoclonium as the Genus
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